Project Summary Recruitment of eosinophils (Eos) from the bloodstream into tissues can occur under a variety of conditions and lead to the release of preformed and newly synthesized products, including cytokines, chemokines, lipid mediators and cytotoxic granule proteins, which can initiate and quickly escalate local inflammatory and remodeling responses. Notably, eosinophil-targeted therapy has been very effective in clinical trials at reducing mature eosinophils in the blood. However, tissue eosinophilia is only partially suppressed and likely results from accessory pathways that continue to promote Eos recruitment and survival. The residual tissue eosinophilia contributes to persistent symptoms and increased risk for tissue damage in patients with Eos-mediated diseases. Thus, new therapies designed with an improved understanding of the mechanism of tissue eosinophilia are needed and likely to have a significant clinical impact. Our preliminary studies implicate Aiolos as a potential regulator of Eos accumulation in eosinophilic asthma as Aiolos-deficiency results in impaired responses to CCR3 ligands and to IL-5, a key cytokine in the pathogenesis of eosinophilic asthma. The central hypothesis of this proposal is that Aiolos controls airway eosinophilia in asthma by two processes, 1) positive regulation of Ccr3 expression by direct transcriptional activation, and 2) a positive feedback loop involving Aiolos, Il5ra, and IL-5. In Aim 1, we will identify the Aiolos-dependent transcriptome in Eos and delineate the mechanism for Aiolos-dependent expression of CCR3. We will also determine the consequence of Aiolos deficiency on Eos-mediated tissue pathology in experimental asthma. In Aim 2, we will determine the mechanism for Aiolos-mediated regulation of IL-5-responsiveness by evaluating the consequence of Aiolos deficiency, haploinsufficiency and overexpression on stage-specific responses by EoPs, eosinophil precursors (preEos) and mature Eos to IL-5. We will also dissect the relationship between Aiolos expression, Il5ra expression and IL-5 stimulation during Eos development in the setting of experimental asthma. Finally, in Aim 3, we will determine the relationship between expression levels of Aiolos in human Eos and 1) a specific Eos gene signature, 2) functional response of Eos to IL-5 and CCL11, and 3) asthma disease severity and Eos phenotype. The high prevalence of eosinophilic inflammation in pediatric asthma and the recent FDA approval of IL-5-targeted therapy for eosinophilic asthma highlight the significance of this application which focuses on delineating the mechanistic relationship between Aiolos expression in Eos, Eos recruitment into the inflamed lung, and IL-5 responsiveness of Eos. Our proposed mechanistic studies using both mouse and human cell systems (Aims 1 and 2) supported by translational studies with Eos from patients with eosinophilic asthma (Aim 3) will provide compelling evidence to support our central hypothesis. The immediate significance of our study is its potential to uncover a dose-dependent relationship between Aiolos expression in Eos and response to IL-5 which would provide rationale for therapy selection for patients based on the Eos phenotype (e.g. Aiolos expression level).